Mechanistic studies on Compound I and II formation in the dye-decolourising peroxidases from Streptomyces lividans

Iron-containing enzymes can utilise oxygen to carry out a variety of oxidative transformations. In the case of haem peroxidases, their interaction with hydrogen peroxide leads to the formation of two FeIV-oxo intermediates: Compound I and Compound II, which are the oxidising species in the catalytic cycle. Dye decolourising peroxidases (DyPs) are the most recent family of haem peroxidases to be discovered and are now known to be widely distributed in bacterial and fungal genomes. Based on phylogenetic and structural analysis, DyPs have been placed into three sub-families (types A, B, C/D), which differ predominately in their cellular location, structure and enzymatic activity. This thesis is focused on three DyPs present in the soil-dwelling bacterium Streptomyces lividans: two A-types, DtpA and DtpAa and a B-type, DtpB. Detailed structural and kinetic studies are reported aimed towards deciphering mechanistic intricacies of Compound I and II formation amongst these DyPs. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV=O) haem species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis combined with the redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal haem pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis.